The present invention is generally in the field of diagnostic and therapeutic reagents, and especially relates to a naturally occurring, circulating soluble alternatively spliced form of human tissue factor (alt-hTF) implicated in thrombotic conditions.
Tissue factor (TF) is the primary initiator of blood coagulation. At sites of vascular injury, formation of a TF:FVIIa complex leads to the generation of FXa, thrombin and the deposition of fibrin to limit hemorrhage. In contrast to its beneficial role in hemostasis, TF initiates life-threatening intravascular thrombosis in sepsis, atherosclerosis and cancer. More recently, TF has been proposed to play a role in other biological processes, including tumor-associated angiogenesis, metastasis and inflammation.
In combination with phospholipid vesicles, TF is used commercially in diagnostic clotting assays. It has always been thought that TF is present in only very small amounts on the surface of cells in the body and not in a circulating form. Before a recombinant source was available, laboratories used thromboplastin, an extract of human brain, placenta or rabbit brain and lung that was previously acetone extracted. The material was lyophilized and resuspended in buffered saline. Until cloned, the structure, exact molecular weight, role of carbohydrate, and relationship with other proteins in humans and other species were not known. Once cloned, the structure could be determined and large amounts of the membrane bound form of human tissue factor (263 amino acids in length; GenBank accession number J02931) or human tissue factor modified to yield a truncated or soluble tissue factor (between 218 and 243 amino acids in length) could be expressed in bacteria and mammalian cells. This tissue factor is used in commercial clotting assays. The soluble tissue factor has the advantage that it is easier to produce, purify and resuspend, as compared to the membrane bound form.
A shorter form of human tissue factor, consisting of amino acid residues 2–219 (Morrissey et al., Blood 1993; 81: 734–744) or 1–218 (Wildgoose et al., Blood 1992; 80: 25–28), produced by recombinant techniques and expression in bacteria, has been reported to be useful in an assay distinguishing between clotting factor VII and activated factor VIIa, when measured in the presence of high quantities of phospholipid.
In blood vessels of healthy humans, tissue factor is found primarily in the adventitia and thus physically separated from coagulation factors, which mainly circulate in an inactive form. Following injury, TF is exposed to blood and initiates the coagulation cascade. The resulting fibrin formation is essential for the initial repair of vessel damage to minimize blood-loss. Therefore, TF may be considered to form a hemostatic sheath around blood vessels essential for hemostasis and appears to be essential for life inasmuch as no TF deficiency has been reported and TF knockout mice do not survive beyond the perinatal period.
TF also plays a crucial role in pathological situations such as coronary artery disease or deep vein thrombosis (DVT). In the former, atherosclerosis is the underlying process leading to pathological disturbances of the arterial wall. The mechanism of venous thrombosis is poorly understood but perhaps blood-borne TF is involved, as reported by Giesen et al., Proc. Natl. Acad. Sci. USA 1999; 96: 2311–2315. Atheromae contain TF as judged by direct bioassay of excised lesions and by immunohistochemistry. Monocytes/macrophages are generally believed to be the major source of this TF although smooth muscle cells near experimental arterial injury contain TF. Upon plaque rupture, TF is exposed to flowing blood thereby allowing circulating factor VII/VIIa to complex with TF. This complex is the catalyst that initiates blood coagulation and thrombosis. However, the deposition of platelets on a TF-coated disc has been reported to inhibit this surface-bound TF, thus implicating circulating TF as necessary for thrombus propagation (Hathcock and Nemerson, Abstract OC2404, Thrombosis and Haemostasis, Supplement, 2001).
The etiology in deep vein thrombosis (DVT) is less well understood. One view of post-surgical thrombosis holds that the procedure causes exposure of TF and perhaps to its release into the blood stream. Combined with patient immobilization this could result in increased risk of thrombosis. Recently, the first clinical trial investigating the efficacy of inhibition of TF activity in prevention of DVT in post-orthopedic surgery patients, has given very promising results. In a open-label, phase II study, the nematode anticoagulant protein NAPc2, which prevents activation of FX by binding to the TF:VIIa:X complex, resulted in a 50% reduction of DVT (Agnelli et al. Blood 2000;96: 491, abstract). This is the first clinical trial that confirms that TF activity plays a critical role in the mechanism of DVT.
It appears that soluble hTF may play a role in these disorders as well as normal coagulation, although its role is not clear. It is also not clear what form this protein may have, or its source. Previously reported functional mutants of human tissue factor have been truncation mutants, typically 1, 2, or 3–218/219, or mutants engineered to contain amino acid substitutions. One commercially available diagnostic reagent is truncated at residue 243.
It is therefore an object of the present invention to provide a method and reagents for diagnosis and treatment of thrombotic conditions based on TF.
It is a further object of the present invention to provide antibodies and other reagents for the detection of naturally occurring circulating soluble hTF.
It is a still further object of the present invention to provide reagents based on the discovery of a circulating soluble alt-hTF, for use as a diagnostic or pharmaceutical.